Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia and is a Category A agent. F. tularensis survives and replicates within macrophages, a phenotype that correlates with virulence, yet little is understood about the molecular basis of its pathogenesis. The long-term goal of this project is to understand how F. tularensis subverts host defenses. This aim will fundamentally interface with the goal of this Program Project, which is to understand how diverse intracellular pathogens such as Mycobacterium tuberculosis, Listeria monocytogenes and Histoplasma capsulatum manipulate host innate responses. We hypothesize that a comparative analysis of the macrophage transcriptional responses to these four intracellular pathogens will be a very powerful means of dissecting and understanding the results of our microarray experiments. We hypothesize that F. tularensis produces gene products that alter the trafficking and perhaps acidification of the initial bacterial phagosome, thereby allowing bacteria to escape into the cytoplasm where it can survive and replicate. The objective of this proposal is to use molecular genetic approaches to uncover F. tularensis molecules that are required for intracellular survival and growth. In a parallel approach, DNA microarrays will be used to characterize the macrophage response to F. tularensis and to test our hypothesis that this pathogen will induce a cytosolic pathway of host gene expression similar to L. monocytogenes. Our specific aims are to 1) identify F. tularensis genes that are expressed inside of macrophages and construct mutants defective in macrophage replication; 2) analyze the intracellular replication and trafficking of F. tularensis; and 3) characterize the macrophage transcriptional response to F. tularensis. These studies will identify regulatory circuits in host cells that are manipulated by F. tularensis and other key intracellular pathogens relevant to biodefense.